The invention relates to a cellfree extract preparation of Haemophilus parasuis, which has toxic activity. The preparation is useful as a vaccine and as a diagnostic agent for Haemophilus parasuis. 
Glxc3xa4sser observed in 1910 a small Gram-negative organism associated with a fibrinous serositis and polyarthritis in swine, which was eventually identified as H. parasuis and distinguished taxonomically from H. suis. It is now accepted that H. parasuis is the infectious agent of porcine polyserositis and arthritis (or Glasser""s disease).
H. parasuis (Hps) is a small pleomorphic Gram-negative rod, varying from a coccobacillary form to slender filaments. Growth is supported only on media containing nicotinamide-adenine dinucleotide (NAD) (heated blood agar, Levinthal agar) or on blood agar in the vicinity of a streak of a Staphylococcus strain (satellitism). Visible growth occurs generally after 36-48 hours incubation.
Porcine polyserositis-arthritis is found worldwide and was historically considered to be a sporadic stress-associated disease of young pigs. More recently, introduction of the pathogen into large populations of specific pathogen free (SPF) herds has shown a devastating effect: infection may spread as a contagious disease of high morbidity, affecting pigs of all ages without obvious associated stress factors. The disease most often affects young pigs (2 weeks to 4 months), principally after the weaning period (5-8 weeks). Mortality may reach 50%.
Serological studies using extracts from autoclaved cells with an agar-gel precipitation test have shown at least fifteen distinct serotypes that differ in their pathogenicity (Kielstein, J. Clin. Micro. 30:4:862, (1992)).
Vaccines for H. parasuis are commercially available, which are made of inactivated bacteria, or bacterins. A disadvantage of bacterin vaccines is that they elicit antibodies against primarily (lipo)polysaccharides that are only specific for a certain serotype of H. parasuis and hence are not protective against other H. parasuis serotypes. The degree of protection against field infection afforded by bacterin vaccines may also be lower than desired.
Even though not currently available, live attenuated H. parasuis vaccines would suffer from the drawbacks normally associated with live vacines, including the risk of inoculating animals with inadequately attenuated pathogens and the possibility that the attenuated bacteria may revert to a pathogenic state resulting in disease in the inoculated animal and spread of the pathogens to other animals.
Thus, there is a need for a H. parasuis vaccine which is safe, effective and provides heterologous protection (serotype independent). To this end, the present inventors endeavored to develop an alternative vaccine.
The rapid onset of disease and severity of internal damage suggest that the clinical effects may be caused by the expression of one or more Hps toxin(s). It has been suggested that Hps may induce functional and structural damage to the nasal mucosa by initially associating with the mucous layer and inducing damage to the underlying mucosa by release of one or more toxic compounds (Vahle, J. L., Ph.D. thesis, AAI9626072, 960902, Iowa State University (1996)). The chemical nature of any such toxins and how they exerted their effects was not elucidated.
Gram-negative endotoxin has been studied as a possible virulence factor of H. parasuis. Endotoxin is a lipopolysaccharide (LPS) component of the outer membrane of Gram-negative bacteria and is released with bacterial lysis. The study of Amano et al (J. Vet. Med. Sci. Vol. 59, No. 6, 451-455 (1997)) reported that Gram-negative endotoxin levels increased in the blood of pigs with acute septicemia caused by H. parasuis, and that H. parasuis antigen was found associated with pathological lesions. However, one cannot conclude on the basis of the findings of Amano et al. that it is the endotoxin that is causing the clinical and pathological manifestations of the disease, and the identity of the factor(s) causing the pathological effects of H. parasuis infection remains unclear.
It is an object of the present invention to find one or more toxins that contribute to the pathology of H. parasuis infection. It is also an object of the present invention to develop vaccines containing the one or more toxins. Finally, it is an object of this invention to use this antigenic material as a reagent in a diagnostic test.
The present invention provides a novel cellfree extract of Hemophilus parasuis, which has toxic activity. This extract causes pathological lesions characteristic of H. parasuis infection when administered intraperitoneally to pigs. Thus, the extract appears to contain at least one toxin, which is termed herein HpTx. It is also contemplated that there may be more than one toxin in this extract.
Also provided are vaccines containing the extract, which are effective in providing protection against homologous and heterologous challenge. It is contemplated that vaccines containing the isolated toxin or toxins as the immunogen(s) will provide this same type of protection.
Finally, the extract is useful as a diagnostic tool, and can be used to raise antibodies that can be used as diagnostic reagents to detect H. parasuis antigen, or could be used itself as a diagnostic reagent to detect antibodies to H. parasuis. Kits for diagnostic tests using these materials are also considered part of the present invention.